To evaluate the duration of the Nfa1-specific IgG antibody responses, the Nfa1-specific IgG antibody formation were evaluated by ELISA in each group at 2, 6 and 12 weeks following third immunization.
In intraperitoneally immunized mice, sera from rNfa1+adjuvant immunized mice demonstrated significantly high levels of the rNfa1-specific IgG at 2 weeks (Fig. 11). These high levels was continued and slightly decreased with 6 and 12 weeks post immunization.
But, during 12 weeks, the value of O.D. of the rNfa1-specific IgG showed significantly high levels compared to normal mice.
In intranasally immunized mice, sera from rNfa1 only immunized mice demonstrated high significantly levels of the rNfa1-specific IgG which was similar to mouse immunized with Nfa1 protein intraperitoneally (Fig. 11).
The duration of the Nfa1-specific IgG levels of mice immunized intraperitoneally with the rNfa1 protein mixed with Freund’s complete adjuvant showed similar to the mice immunized intranasally with the rNfa1 protein only.
Fig. 11. Duration of the Nfa1-specific IgG levels in immune mice sera. The Nfa1-specific IgG responses were evaluated by ELISA in each group at 2, 6 and 12 weeks following immunization.
0 0.5 1 1.5 2 2.5 3
2 6 12
Weeks
O .D .( 4 0 5 n m )
Group Ⅶ Ⅶ Ⅶ, Normal (PBS) (i.p.) Ⅶ Group Ⅷ Ⅷ Ⅷ Ⅷ, rNfa1+Adjuvant (i.p.)
Group Ⅸ Ⅸ Ⅸ, Normal (PBS) (i.n.) Ⅸ Group Ⅹ Ⅹ Ⅹ Ⅹ, rNfa1 (i.n.)
Ⅳ Ⅳ
Ⅳ Ⅳ. DISCUSSION
N. fowleri is the causal agent of primary amoebic meningoencephalitis (PAME).
Naegleria spp. are amoeboflagellates found in water and soil. Although, some 30 species of Naegleria have been recognized based upon sequencing data (De Jonckheere, 2004), N.
fowleri is the only one that has been isolated from cases of primary amoebic meningoencephalitis. Other species (Naegleria italica, Naegleria philippinensis, Naegleria australiensis) may be pathogenic in the mouse model of PAME, but have not been identified from any human cases.
PAME is a fulminating disease, developing within several days following exposure to the water source, and causing death within 1~2 weeks after hospitalization. Adherence of pathogens to host cells is a critical initial step in the infection process. The ability of trophozoites to attach to the nasal mucosal, an increased rate of locomotion, and a chemotactic response to nerve cell components may be important in disease progression (Cline et al., 1986; Brinkley and Marciano-Cabral, 1992; Han et al., 2004).
A variety of in vitro cell culture systems have been used to study the infection of N.
fowleri with mammalian cells. N. fowleri trophozoites have been shown to destroy nerve cells, as well as other cell types, by trogocytosis using food-cup structure on their surface (Brown, 1979; Marciano-Cabral et al., 1982; Marciano-Cabral and Fulford, 1986) and by the release of cytolytic molecules (Lowery and McLaughlin, 1984, 1985; Marciano-Cabral and Fulford, 1986; Leippe and Herbst, 2004).
Many antiparasitic and antimicrobial drugs have been screened for in vitro and in vivo activity against N. fowleri. Naegleria spp. are highly sensitive to the antifungal drug amphotericin B, and it has been used in virtually all cases as the core antimicrobial where recovery occurred. Minimum amoebacidal concentrations of amphotericin B were determined to be 0.02∼0.078 ㎍/ml for three different clinical isolates of N. fowleri tested in vitro (Duma et al., 1971).
Particularly in the early stages of PAME, Naegleria infections have also been treated successfully with amphotericin B, rifampin, and chloramphenicol; amphotericin B, oral rifampin, and oral ketoconazole; and amphotericin B alone (Abramowicz, 2004; Schuster and Visvesvara, 2004; Schuster and Visvesvara, 2004).
Circulating IgG antibodies in serum were demonstrated by ELISA from day 7 after infection in ICR mice infected with N. fowleri (Park et al., 1987b) and from day 14 after infection in ICR mice infected with N. jadini, but no anti-N.gruberi antibody was detected (Lee et al., 1985).
In the present study, the immunogenicity of the rNfa1 protein and protective efficacy for pathogenic N. fowleri infection were evaluated. The survival times of intraperitoneally or intranasally immunized mice with the rNfa1 protein prolonged about 10 or 9 days in comparison with the normal group mice. Mice immunized intraperitoneally with the Nfa1 protein in adjuvant or in the absence of adjuvant, or intranasally with the rNfa1 only elicited significantly high levels of the Nfa1-specific IgG antibody. Furthermore, analysis of IgG subclass profiles revealed that anti-Nfa1 IgG1 showed the greatest increase followed by IgG2b, IgG2a and IgG3, suggesting that rNfa1-adjuvant or only rNfa1 immunization resulted
in a Th1/Th2 mixed type immunity. Also, intraperitoneal or intranasal immunization of Nfa1 showed significantly high levels of the Nfa1-specific serum IgA antibody. It is generally known that mucosal IgA plays an important role in protection against several bacterial, viral and protozoan (Renegar et al., 1991; Michetti et al., 1992; Marcotte et al., 1998). The inhibition of bacterial adherence by mucosal IgA is considered to be one of the most important defense mechanisms against mucosal bacterial invasion, has been shown to limit the attachment of bacteria to epitherial cells (Svanborg-Eden et al., 1978; Kurono Y et al., 1989).
Also, adherence of N. fowleri to host cells is a critical initial step in the infection process. N. fowleri penetrates the mucosal epithelial layer and migrates along the olfactory nerve tracts, crossing the cribriform plate, to the brain. Mice immunized intranasally with antigen-adjuvant showed low levels of serum IgA and high levels of mucosal IgA (Masanori et al., 1998). In this study, mice immunized intraperitoneally with the rNfa1 in adjuvant or in the absence of adjuvant, or intranasally with the rNfa1 only showed significantly high levels of the Nfa1-specific serum IgA. Finally, the survival times were prolonged. It suggests that antibody may act to the resistance to N. fowleri penetration and invasion by immobilizing the N. fowleri and inhibiting the adherence of the N. fowleri to nasal mucosal. Interestingly, IgE responses were not detected in these mice, suggesting that immune responses to the rNfa1 are not mediated by hypersensitivity reactions.
Immunoglobulin isotype-specific responses seen in the rNfa1-adjuvant or only rNfa1 immunized mice are consistent with cytokine profiles in splenocyte culture significant levels of Th1 type cytokine, γ-IFN and IL-2 were demonstrated, together with Th2 type cytokine,
IL-4 levels. Also, immuno regulatory cytokine IL-10 was detected. These data suggest that the rNfa1 triggers a Th1/Th2 mixed type of immune response. It is proposed that a high level of γ-IFN production may suppress IL-4 production by splenocytes in mice immunized with rNfa1-adjuvant or only rNfa1, since γ-IFN and IL-4 affect each other as antagonists (Pene et al., 1988).
Using this mouse model, several attempts have been shown that mice can be immunized against N. fowleri using either living amoeba given by intraperitoneal route or amoeba killed by formalin-fixed or by freezing-thawing and led to diverse results with partial protective immunity (Thong et al., 1978a; 1978b). Also, it has been made to induce protective immunity against N. fowleri, but the study of immunity in experimental infections and PAME patients has been limited to the analysis of serum antibody response (Thong YH et al., 1980; Bush LE and John DT., 1988; Ferrante A., 1991). The present study attempted to help comprehension of protective immunity of mice immunized with Nfa1 protein against N.
fowleri infection and the role of afresh cloned Nfa1 protein was made clear regarding the presence of antigen- or pathogen-related protein and their functions in N. fowleri.
To fix up the lethal dose of the N. fowleri trophozoites, three groups of 5 mice were intranasally inoculated with 1 × 102, 1 × 104, or 1 × 106 N. fowleri trophozoites in preliminary experiment. The infected mice were monitored for up to 30 days. The lethal dose choice was 1 × 104 N. fowleri trophozoites, since this dose killed all infected mice by day 17
after inoculation. Nevertheless, the survival times in mice infected intranasally with N.
fowleri trophozoites post immunization intraperitoneally with the rNfa1 in adjuvant or in the absence of adjuvant, or intranasally with the rNfa1 only were prolonged about 10 or 9 days
compared with the normal group mice.
These data suggest that the rNfa1 protein may induce the humoral and cell-mediated immunity leading to the host defense in N. fowleri infection.
In summary, sera of mice immunized with the rNfa1 protein intraperitoneally or intranasally had a significantly increased level of IgG. Analysis of IgG subclass profiles of mice immunized with the rNfa1 protein intraperitoneally or intranasally revealed that IgG1 showed the greatest increase followed by IgG2b, IgG2a, and IgG3. Splenocytes of mice immunized with the rNfa1 protein intraperitoneally or intranasally secreted significantly high levels of both Th1 cytokine, γ-IFN and Th2 cytokine, IL-10.
These results demonstrate that Nfa1 protein is an effective antigen to improve protective immunity to N. fowleri infection and provide feasibility of Nfa1 as a vaccine for N. fowleri infection and basic features of Nfa1 induced immune responses important for improvement of vaccine efficacy.
Ⅴ Ⅴ
Ⅴ Ⅴ. CONCLUSION
In the present study, to observe the protective immunity for N. fowleri trophozoites infection in a mouse model, BALB/c mice were intraperitoneally or intranasally immunized with the rNfa1 protein. After final booster of the rNfa1 protein, 1 x 104 trophozoites of N.
fowleri were intranasally inoculated into mice.
For intraperitoneally immunization, mice were divided into four groups; Group I, Normal; Group II, Adjuvant+PBS; Group III, rNfa1+Adjuvant; Group IV, rNfa1 only. The mice were immunized intraperitoneally with the rNfa1 protein in adjuvant or in the absence of adjuvant. The survival times of mice in group I, II, III and IV were 15.5, 15.0, 25.0 and 24.4 days, respectively. Sera obtained from mice of the group III and IV had a significantly increased level of the Nfa1-specific IgG. Analysis of the Nfa1-specific IgG subclass profiles revealed that IgG1 showed the greatest increase followed by IgG2b, IgG2a and IgG3.
Splenocytes obtained from intraperitoneally immunization group Ⅲ (rNfa1+Adjuvant) and group IV (rNfa1 only) secreted significantly high levels of both Th1 cytokine, gamma-interferon and Th2 cytokine, interleukin-10 after stimulation with rNfa1 in vitro.
For intranasally immunization, mice were divided into two groups; Group Ⅴ, Normal;
Group Ⅵ, rNfa1 only. The mice were immunized intranasally with the rNfa1 protein only.
The survival times of mice in group Ⅴ, Ⅵ were 15.0 and 24.7 days, respectively. Sera obtained from immune protected mice had a significantly increased level of the Nfa1-specific IgG. Analysis of the Nfa1-Nfa1-specific IgG subclass profiles revealed that IgG1 showed
the greatest increase followed by IgG2b, IgG2a, and IgG3. Splenocytes obtained from intranasally immunization group Ⅵ (rNfa1 only) mice secreted significantly high levels of both Th1 cytokine, gamma-interferon and Th2 cytokine, interleukin-10 after stimulation with rNfa1 in vitro.
In summary the survival times of intraperitoneally or intranasally immunized mice with the rNfa1 protein were prolonged about 10 or 9 days in comparison with the normal group mice, and then the rNfa1-specific antibody formation and rNfa1-induced Th1/Th2-mediated effector mechanisms are required for the protection of N. fowleri infection.
These results suggested that the Nfa1 protein induced the protective immunity in PAME developed mice due to N. fowleri infection.
REFERENCES
1. Abramowicz M: Drugs for parasitic infections. Med Lett Drugs Ther 46: 1∼12, 2004
2. Brinkley C, Marciano-Cabral F: A method for assessing the migratory response of Naegleria fowleri utilizing [3H] uridine-labeled amoebae. J Protozool 39: 297∼303, 1992
3. Brown T: Observations by immunofluorscence microscopy on the cytopathogenicity of Naegleria fowleri in mouse embryo-cell cultures. J Med Microbiol 12: 363∼371, 1979
4. Bush LE, John DT: Intranasal immunization of mice against Naegleria fowleri. J Protozool 35: 172∼176, 1988
5. Cerva L: Acanthamoeba culbertsoni and Naegleria fowleri: occurrence of antibodies in man. J Hyg Epidemiol Microbiol 33: 99∼103, 1989
6. Cline M, Carchman R, Marciano-Cabral F: Movement of Naegleria fowleri stimulated by mammalian cells in vitro. J Protozool 33: 10∼13, 1986
7. Culbertson CG: The pathogenicity of soil amebas. Annu Rev Microbiol 25: 231∼254, 1971
8. Culbertson CG, Smith JW, Minner JR: Acanthamoeba: observations on animal pathogenicity. Science 127: 1506, 1958
9. Cursons RTM, Brown TS, Keys EA: Virulence of pathogenic free-living amoebae. J Parasitol 64: 744∼745, 1978
10. De Jonckheere JF: Molecular definition and the ubiquity of species in the genus Naegleria. Protist 155: 89∼103, 2004
11. De Jonckheere J, van de Voorde H: Comparative study of six strains of Naegleria with special reference to nonpathogenic variants of Naegleria fowleri. J Protozool 24:
304∼309, 1977a
12. De Jonckheere J, van de Voorde H: The distribution of Naegleria fowleri in man-made thermal waters. Am J Trop Med Hyg 26: 10∼15, 1977b
13. Dubray BL, Wilhelm WE, Jennings BR: Serology of Naegleria fowleri and Naegleria lovaniensis from a hospital survey. J Protozool 34: 322∼327, 1987
14. Duma RJ, Rosenblum WI, McGehee RF, Jones MM, Nelson EC: Primary amoebic meningoencephalitis caused by Naegleria. Two new cases, response to amphotericin B, and a review. Ann Int Med 74: 923∼931, 1971
15. Eisen D, Franson RC: Acid-active neuraminidases in growth media from cultures of pathogenic Naegleria fowleri and in sonicates of rabbit alveolar marcrophages. Biochim Biophys Acta 924: 369∼372, 1987
16. Ferrante A: Free-living amoebae: pathogenecity and immunity. Parasite Immunol 13:
31∼47, 1991
17. Fowler M, Cater RF: Acute pyogenic meningitis probably due to Acanthamoeba sp: a preliminary report. Br Med J 2: 740∼743, 1965
18. Goswick SM, Brenner GM: Activities of azithromycin and amphotericin B against Naegleria fowleri in vitro and in a mouse model of primary amebic meningoencephalitis. Antimicrob Agents Chemother 47: 524∼528, 2003a
19. Han KL, Lee HJ, Shin MH, Shin HJ, Im KI, Park SJ: The involvement of an integrin-like protein and protein kinase C in amoebic adhesion to fibronectin and amoebic cytotoxicity.
Parasitol Res 94:53∼60, 2004
20. Jager BV, Stamm WP: Brain abscesses caused by free-living amoeba probably of the genus Hartmannella in a patient with Hodgkin's disease. Lancet 2: 1343∼1345, 1972
21. Jeong SR, Kang SY, Lee SC, Park S, Kim KH, Im KI, Shin HJ: Decreasing effect of an anti-Nfa1 polyclonal antibody on the in vitro cytotoxicity of pathogenic Naegleria fowleri. Korean J Parasitol 42: 35∼40, 2004
22. John DT, Cole Jr TB, Bruner RA: Amebostomes of Naegleria fowleri. J Protozool 32:
12∼19, 1985
23. John DT, John RA: Cytopathogenicity of Naegleria fowleri in mammalian cell cultures.
Parasitol Res 76: 20∼25, 1989
24. John DT, Smith BL: Amebicidal activity of wild animal serum. J Parasitol 83: 757∼759, 1997
25. Jones DB, Visvesvara GS, Robinson NM: Acanthamoeba polyphaga keratitis and Acanthamoeba uveitis associated with fatal meningoencephalitis. Trans Ophthalmol Soc UK 95: 221∼232, 1975
26. Kollars TM, Wilhelm WE: The occurrennce of antibodies to Naegleria species in wild mammals. J Parasitol 82: 73∼77, 1996
27. Kurono Y, Fujiyoshi T, Mogi G: Secretory IgA and bacterial adherence to nasal mucosal cells. Ann Otol Rhinol Laryngol 98: 273∼278, 1989
28. Lares-Villa F, De Jonckheere JF, De Moura H, Recchi-Iruretagoyena A, Ferreira-Guerrero E, Fernandez-Quintanilla G, Ruiz-Matus C, Visvesvara GS: Five cases of primary amebic meningoencephalitis in Mexicali, Mexico: study of the isolates. J Clin Microbiol 31: 685∼688, 1993
29. Lee SG, Im KI, Lee KT: Protective immunity against Naegleria meningoencephalitis in mice. Korean J Parasit 23: 293∼299, 1985
30. Lee YJ, Kim JH, Jeong SR, Song KJ, Kim K, Prak S, Park MS, Shin HJ: Production of Nfa1-specific monoclonal antibodies that influences the in vitro cytotoxicity of Naegleria fowleri trophozoites on microglial cells. Parasitol Res 101: 1191∼1196, 2007
31. Leippe M, Herbst R: Ancient weapons for attack and defense: the pore-forming polypeptides of pathogenic enteric and free-living amoeboid protozoa. J Euk Microbiol 51: 516∼521, 2004
32. Lowrey DM, McLaughlin J: A multicomponent hemolytic system in the pathogenic amoeba Naegleria fowleri. Infect Immun 45: 731∼736, 1984
33. Lowrey DM, McLaughlin J: Activation of a heat-stable cytolytic protein associated with the surface membrane of Naegleria fowleri. Infect Immun 50: 478 ∼482, 1985
34. Marciano-Cabral F: Biology of Naegleria spp. Microbiol Rev 52: 114∼133, 1988
35. Marciano-Cabral F, Cline ML, Bradley SG: Specificity of antibodies from human sera for Naegleria species. J Clin Microbiol 25: 692∼697, 1987
36. Marciano-Cabral F, Fulford DE: Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells. Appl Environ Microbiol 51:
1133∼1137, 1986
37. Marciano-Cabral F, Patterson M, John DT, Bradley SG: Cytopathogenicity of Naegleria fowleri and Naegleria gruberi for established mammalian cell cultures. J Parasitol 68:
1110∼1116, 1982
38. Marcotte H, Lavoie MC: Oral microbial ecology and the role of salivary immunoglobulin A. Microbiol Mol Biol Rev 62: 71∼109, 1998
39. Martinez AJ: Is Acanthamoeba encephalitis an opportunistic infection? Neurology 30:
567∼574, 1980
40. Martinez AJ: Free-Living Amebas: Natural History, Prevention, Diagnosis, Pathology, and Treatment of Disease. CRC Press Boca Raton Florida 156, 1985
41. Masanori I, Yoko T, Satoshi K, Yutaka M, Tooru T, Keiko M, Kunio T: Systemic and mucosal immune responses of mice to aluminium-adsorbed or aluminium-non-adsorbed tetanus toxoid administered intranasally with recombinant cholera toxin B subunit.
Vaccine 16: 1620∼1626, 1998
42. Michetti P, Mahan MJ, Slauch JM, Mekalanos JJ, Neutra MR: Monoclonal secretory immunoglobulin A protects mice against oral challenge with the invasive pathogen Salmonella tryphimurium. Infect Immun 60: 1786∼1792, 1992
43. Naginton J, Watson PG, Playfair TJ, McGill J, John BR, Steele ADMcG: Amoebic infection of the eye. Lancet 2: 1537∼1540, 1974
44. Page FC: Rosculus ithacus Hawes, 1963 (Amoebida, Flabellidae) and the amphizoic tendency in amoebae. Acta Protozool 13: 143∼154, 1974
45. Park KM, Ryu JS, Im KI: Blastogenic responses of splenic lymphocytes to Naegleria fowleri lysates and T-cell mitogen in mice with primary amoebic meningoencephalitis.
Korean J Parasit 25: 1∼6, 1987b
46. Renegar KB, Small Jr PA: Passive transfer of local immunity to influenza virus infection by IgA antibody. J Immunol 146: 19721∼978, 1991
47. Schuster FL: Cultivation of pathogenic and opportunistic free-living amebas. Clin Microbiol Rev 15: 342∼354, 2002
48. Schuster FL, Dunnebacke TH, Visvesvara GC: Activity of selected antimicrobials against clinical isolates of pathogenic free-living amebas. John Libbey Eurotext 45∼48, 2001
49. Schuster FL, Rechthand E: In vitro effects of amphotericin B on growth and ultrastructure of the amoeboflagellates Naegleria gruberi and Naegleria fowleri.
Antimicrob Agents Chemother 8: 591∼605, 1975
50. Schuster FL, Visvesvara GS: Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int J Parasitol 34: 1001∼1027, 2004
51. Seidel JS, Harmatz P, Visvesvara GS, Cohen A, Edwards J, Turner J: Successful treatment of primary amoebic meningoencephalitis. N Engl J Med 306: 346∼348, 1982
52. Shin HJ, Cho MS, Jung SY, Kim HI, Park S, Im KI: Molecular cloning and
characterization of a gene encoding a 13.1 kDa antigenic protein of Naegleria fowleri. J Euk Microbiol 48: 713∼717, 2001
53. Stevens AR, De Jonckheere J, Willaert E: Naegleria lovaniensis new species: Isolation and identification of six thermophilic strains of a new species found in association with Naegleria fowleri. Int J Parasitol 10: 51∼64, 1980
54. Svanborg-Eden C, Svennerholm AM: Secretory immunoglobulin A and G antibodies prevent adhesion of Escherichia coli to human urinary epitherial cells. Infect Immun 22:
790∼797, 1978
55. Thong YH, Ferrante A, Rowan-Kelly B, O'Keefe DE: Immunization with live amoebae, amoebic lysate and culture supernatant in experimental Naegleria meningoencephalitis.
Trans Roy Soc Trop Med Hyg 74: 570∼576, 1980
56. Thong YH, Ferrante A, Shepherd D, Rowan-Kelly B: Resistance of mice to Naegleria meningoencephalitis transferred by immune serum. Trans R Soc Trop Med Hyg 72: 650, 1978a
57. Thong YH, Shepherd C, Ferrante A, Rowan-Kelly B: Protective immunity to Naegleria fowleri in experimental amoebic meningoencephalitis. Amer J Trop Med 27: 238, 1978b
58. Visvesvara GS: Pathogenic and opportunistic free-living amebae. Manual of Clinical Microbiology 7th Edition ASM Press, pp.1383∼1390, 1999
59. Visvesvara GS, Schuster FL, Martinez AJ: Balamuyhia mandrillaris, N.G., N. Sp., agent of amebic meningoencephalitis in humans and other animals. J Euk Microbiol
40: 504∼514, 1993
60. Visvesvara GS, Stehr-Green JK: Epidemiology of free-living amoeba infections. J Protozool 37 (suppl) 25∼33, 1990
61. Whiteman LY, Marciano-Cabral F: Susceptibility of pathogenic and nonpathogenic Naegleria spp. to complement-mediated lysis. Infect Immun 55: 2442∼2447, 1987
62. Wong MM, KarrSL, Chow CK: Changes in the virulence of Naegleria fowleri maintained in vitro. J Parasitol 63: 872∼878, 1977
63. Young JDE, Lowrey DM: Biochemical and functional characterization of a membrane- associated pore-forming protein from the pathogenic ameboflagellate Naegleria fowleri.
J Biol Chem 264: 1077∼1083, 1989
amoebic meningo-encephalitis)을 유발하여 7~14일 이내에 죽음에 이르게 하는 것으 로 알려져 있다.
우스의 평균 사망일이 25.0일과 24.4일이었다. 각 그룹의 마우스 마우스에서 얻은 혈청 에서 immunoglobulin G (IgG)의 수치와 IgG subclass 수치를 측정한 결과, 그룹Ⅲ에서 가장 높았으며, IgG1, IgG2b, IgG2a, IgG3순이었다. In vitro 실험에서, 각 그룹의 마우스 로부터 비장세포를 분리한 후, rNfa1 단백질로 자극을 주고 cytokine 분비량을 측정한 결과, 그룹Ⅲ의 마우스 비장세포에서 특히 Th1 cytokine인 γ-IFN 와 Th2 cytokine 인 IL-10이 높은 수치를 보였다. 다른 한편으로, 복강이 아닌 다른 방법으로 면역하기 위해서 그룹Ⅴ (대조군), 그룹Ⅵ (rNfa1)로 나누었다. 각각의 마우스에 비강으로 면역을
우스의 평균 사망일이 25.0일과 24.4일이었다. 각 그룹의 마우스 마우스에서 얻은 혈청 에서 immunoglobulin G (IgG)의 수치와 IgG subclass 수치를 측정한 결과, 그룹Ⅲ에서 가장 높았으며, IgG1, IgG2b, IgG2a, IgG3순이었다. In vitro 실험에서, 각 그룹의 마우스 로부터 비장세포를 분리한 후, rNfa1 단백질로 자극을 주고 cytokine 분비량을 측정한 결과, 그룹Ⅲ의 마우스 비장세포에서 특히 Th1 cytokine인 γ-IFN 와 Th2 cytokine 인 IL-10이 높은 수치를 보였다. 다른 한편으로, 복강이 아닌 다른 방법으로 면역하기 위해서 그룹Ⅴ (대조군), 그룹Ⅵ (rNfa1)로 나누었다. 각각의 마우스에 비강으로 면역을